Alexandra M. Dedman

A Sphingosine-1–Phosphate-Activated Calcium Channel Controlling Vascular Smooth Muscle Cell Motility

In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1–phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5–TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation.

Sensing of Lysophospholipids by TRPC5 Calcium Channel

TRPC calcium channels are emerging as a ubiquitous feature of vertebrate cells, but understanding of them is hampered by limited knowledge of the mechanisms of activation and identity of endogenous regulators. We have revealed that one of the TRPC channels, TRPC5, is strongly activated by common endogenous lysophospholipids including lysophosphatidylcholine (LPC) but, by contrast, not arachidonic acid. Although TRPC5 was stimulated by agonists at G-protein-coupled receptors, TRPC5 activation by LPC occurred downstream and independently of G-protein signaling. The effect was not due to the generation of reactive oxygen species or because of a detergent effect of LPC. LPC activated TRPC5 when applied to excised membrane patches and thus has a relatively direct action on the channel structure, either because of a phospholipid binding site on the channel or because of sensitivity of the channel to perturbation of the bilayer by certain lipids. Activation showed dependence on side-chain length and the chemical head-group. The data revealed a previously unrecognized lysophospholipid-sensing capability of TRPC5 that confers the property of a lipid ionotropic receptor.

Discrete store‐operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle

This study tested the hypothesis that store‐operated channels (SOCs) exist as a discrete population of Ca2+ channels activated by depletion of intracellular Ca2+ stores in cerebral arteriolar smooth muscle cells and explored their direct contractile function. Using the Ca2+ indicator fura‐PE3 it was observed that depletion of sarcoplasmic reticulum (SR) Ca2+ by inhibition of SR Ca2+‐ATPase (SERCA) led to sustained elevation of [Ca2+]i that depended on extracellular Ca2+ and slightly enhanced Mn2+ entry. Enhanced background Ca2+ influx did not explain the raised [Ca2+]i in response to SERCA inhibitors because it had marked gadolinium (Gd3+) sensitivity, which background pathways did not. Effects were not secondary to changes in membrane potential. Thus SR Ca2+ depletion activated SOCs. Strikingly, SOC‐mediated Ca2+ influx did not evoke constriction of the arterioles, which were in a resting state. This was despite the fura‐PE3‐indicated [Ca2+]i rise being greater than that evoked by 20 mm[K+]o (which did cause constriction). Release of endothelial vasodilators did not explain the absence of SOC‐mediated constriction, nor did a change in Ca2+ sensitivity of the contractile proteins. We suggest SOCs are a discrete subset of Ca2+ channels allowing Ca2+ influx into a ‘non‐contractile’ compartment in cerebral arteriolar smooth muscle cells.

Expression and function of native potassium channel (KVα1) subunits in terminal arterioles of rabbit

1 In this study we investigated the expression and function of the KVα1 subfamily of voltage‐gated K+ channels in terminal arterioles from rabbit cerebral circulation. 2 K+ current was measured from smooth muscle cells within intact freshly isolated arteriolar fragments. Current activated on depolarisation positive of about –45 mV and a large fraction of this current was blocked by 3,4‐diaminopyridine (3,4‐DAP) or 4‐aminopyridine (4‐AP), inhibitors of KV channels. Expression of cRNA encoding KV1.6 in Xenopus oocytes also generated a 4‐AP‐sensitive K+ current with a threshold for activation near –45 mV. 3 Immunofluorescence labelling revealed KV1.2 to be specifically localised to endothelial cells, and KV1.5 and KV1.6 to plasma membranes of smooth muscle cells. 4 KV channel current in arteriolar fragments was blocked by correolide (which is specific for the KVα1 family of KV channels) but was resistant to recombinant agitoxin‐2 (rAgTX2; which inhibits KV1.6 but not KV1.5). Heterologously expressed KV2.1 was resistant to correolide, and KV1.6 was blocked by rAgTX2. 5 Arterioles that were mildly preconstricted and depolarised by 0.1–0.3 nm endothelin‐1 constricted further in response to 3,4‐DAP, 4‐AP or correolide, but not to rAgTX2. 6 We suggest that KVα1 channels are expressed in smooth muscle cells of terminal arterioles, underlie a major part of the voltage‐dependent K+ current, and have a physiological function to oppose vasoconstriction. KVα1 complexes without KV1.5 appear to be uncommon.

TRPC channel lipid specificity and mechanisms of lipid regulation.

TRPC channels are a subset of the transient receptor potential (TRP) proteins widely expressed in mammalian cells. They are thought to be primarily involved in determining calcium or sodium entry and have broad-ranging functions that include regulation of cell proliferation, motility and contraction. The channels do not respond to a single stimulator but rather are activated or modulated by a multiplicity of factors, potentially existing as integrators at the plasma membrane. This review considers the sensitivity of TRPCs to lipid factors, with focus on sensitivities to diacylglycerols, lysophospholipids, arachidonic acid and its metabolites, sphingosine-1-phosphate (S1P), cholesterol and derivatives, and other lipid factors such as gangliosides. Promiscuous and selective lipid-sensing are apparent. In many cases the lipids stimulate channel function or increase insertion of channels in the membrane. Both direct and indirect (receptor-dependent) lipid effects are evident. Although information is limited, the lipid profiles are consistent with TRPCs having close working relationships with phospholipase C and A2 enzymes. We need much more information about lipid-sensing by TRPCs if we are to fully appreciate its significance, but the available data suggest that lipid-sensing is a key, but not exclusive, aspect of TRPC biology.

Activation thresholds of K(V), BK andCl(Ca) channels in smooth muscle cells in pial precapillary arterioles.

We have previously shown expression of voltage-gated K⁺ channels (K_V) in smooth muscle of cerebral arterioles and suggested the channels function to oppose voltage-dependent Ca²⁺ entry. However, other studies indicate that large conductance Ca²⁺-activated K⁺ (BK) channels serve this function and chloride (Cl^–) channels may have the opposite effect. In this study we compared the activation thresholds and absolute current amplitudes for K_V channels, BK channels and Cl^– channels at physiological membrane potentials in intact precapillary arterioles from the rabbit cerebral circulation. Patch-clamp recordings were made to measure current and membrane potential, and a video scan line was used to detect external diameter. Two strategies to determine the basal current-voltage relationship of BKchannels showed the channels contributed current only at voltages positive of –35 mV, even though voltage-dependent Ca²⁺-entry occurred. Ca²⁺-activated and niflumic acid-sensitive Cl^– current was detected but, between –50 and –10 mV, both BK and Cl^– channel currents were much smaller and contributed less to the membrane potential compared with K_V channel current. Furthermore, in the absence of an exogenous vasoconstrictor agent, block of K_V channels but not BK or Cl^– channels caused constriction, although in the presence of endothelin-1 block of BK or K_V channels caused constriction. The data indicate K_V channels are the first inhibitory mechanism to activate when there is depolarisation in precapillary arteriolar smooth muscle cells of the cerebral circulation.

Modulation of TRPC5 cation channels by halothane, chloroform and propofol

TRPC5 is a mammalian homologue of the Drosophila Transient Receptor Potential (TRP) channel and has expression and functions in the cardiovascular and nervous systems. It forms a calcium‐permeable cation channel that can be activated by a variety of signals including carbachol (acting at muscarinic receptors), lanthanides (e.g. Gd3+) and phospholipids (e.g. lysophosphatidylcholine: LPC). Here we report the effects of inhalational (halothane and chloroform) and intravenous (propofol) general anaesthetics upon TRPC5.

TRPC1 transcript variants, inefficient nonsense- mediated decay and low up-frameshift-1 in vascular smooth muscle cells

BackgroundTransient Receptor Potential Canonical 1 (TRPC1) is a widely-expressed mammalian cationic channel with functional effects that include stimulation of cardiovascular remodelling. The initial aim of this study was to investigate variation in TRPC1-encoding gene transcripts.ResultsExtensive TRPC1 transcript alternative splicing was observed, with exons 2, 3 and 5-9 frequently omitted, leading to variants containing premature termination codons. Consistent with the predicted sensitivity of such variants to nonsense-mediated decay (NMD) the variants were increased by cycloheximide. However it was notable that control of the variants by NMD was prominent in human embryonic kidney 293 cells but not human vascular smooth muscle cells. The cellular difference was attributed in part to a critical protein in NMD, up-frameshift-1 (UPF1), which was found to have low abundance in the vascular cells. Rescue of UPF1 by expression of exogenous UPF1 was found to suppress vascular smooth muscle cell proliferation.ConclusionsThe data suggest: (i) extensive NMD-sensitive transcripts of TRPC1; (ii) inefficient clearance of aberrant transcripts and enhanced proliferation of vascular smooth muscle cells in part because of low UPF1 expression.